This project is exploring the organization of NADPH oxiase genes. This enzyme is responsible for production of superoxide and related microbicidal oxidants. Deficiencies in this host defense system result in chronic granulomatous disease (CGD), which is characterized by serious recurrent infections and excess granuloma formation. This program will explore genetic lesions that affect NADPH oxidase activity in CGD, provide gene mapping information enabling diagnosis of CGD, and suggest strategies for correction of these gene defects. Current efforts are focused on the gene encoding the p67-phox oxidase component. Genomic clones from this locus have been extensively characterized with respect to all exon-intron boundaries and their flanking sequences. This information has enabled design of oligonucleotide primers needed for PCR analysis of CGD lesions using small amounts of genomic DNA from any tissue source. Using this method, a 20 basepair deletion on the 3' boundary of exon 5 was noted in one p67-phox deficient patient. The corresponding cDNA from this patient was cloned to define the effects of this abberrant splice site, which likely resulted from an uneven crossover event. Several other p67-phox deficient patients have been identified and several structural variations (deletions, point mutations, and insertions) have been observed. A related project (Z01 AI 00614) has established the means for stable gene transfection and oxidase restoration using episomal vectors that endow hygromycin resistance. The effects of these putative CGD gene defects on NADPH oxidase activity will be explored with this gene transfer system. We are developing this gene transfer method to produce stable "model CGD lines", which express three of the four oxidase components and will enable functional analysis of lesions in any of the four CGD genes.